Chitin-binding proteins of Artemia diapause cysts participate in formation of the embryonic cuticle layer of cyst shells
Chitin-binding proteins of Artemia diapause cysts participate in formation of the embryonic cuticle layer of cyst shells
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DOI：
10.1042/bj20121259
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发表时间：
2013-01-01
影响因子：
4.1
通讯作者：
Yang, Wei-Jun
中科院分区：
文献类型：
--
作者：
Ma, Wen-Ming;Li, Hua-Wei;Yang, Wei-Jun
贫血虾要么通过卵胎生繁殖，产生自由游动的无鳞虾，要么通过卵胎生繁殖，产生能够适应恶劣和复杂栖息地的囊状胚胎（滞育囊肿）。当包囊进入滞育时，它们被包裹在一个复杂的外壳中，保护它们免受某些极端环境的影响。卵生和卵胎生卵卵的基因组比较以前已经描述过。我们从卵生卵母细胞中分离到3个显著上调的基因，鉴定为Arp-CBP （Anemia parthenogenetica几丁质结合蛋白）基因。实时荧光定量PCR结果显示，在滞育囊肿形成过程中，Arp-CBP基因的表达逐渐增加，在卵生发育的卵囊阶段，mRNA的积累显著。此外，原位杂交结果表明，在胚胎中表达了Arp-CBP mrna。有趣的是，免疫电镜结果显示，这三种Arp-CBPs都分布在囊肿壳的细胞ECL（胚胎角质层）中。此外，通过RNA干扰敲低Arp-CBP导致胚胎角质层组成发生显著变化。抑制Arp-CBP表达后，囊壳纤维层呈松散形态，内、外角质层呈明显不规则。最后，体外重组蛋白结合实验表明，这三种Arp-CBPs都具有碳水化合物结合活性。这些发现为揭示青蒿囊壳ECL的形成机制提供了重要的见解，并证明了Arp-CBPs参与了纤维晶格的构建，并且是囊壳ECL形成所必需的。
The brine shrimp Anemia reproduces either ovoviviparously, producing free-swimming nauplii, or oviparously, producing encysted embryos (diapause cysts) able to cope with harsh and complex habitats. When the cysts enter diapause they are encased in a complex external shell that protects them from certain extreme environments. The genomic comparison of oviparous and ovoviviparous ovisacs has been described previously. We isolated three significantly up-regulated genes in oviparous oocytes and identified them as Arp-CBP (Anemia parthenogenetica chitin-binding protein) genes. Quantitative real-time PCR indicated that the expression of Arp-CBP genes gradually increases during diapause cyst formation and significant mRNA accumulation occurs during the ovisac stage of oviparous development. Moreover, in situ hybridization results demonstrated that Arp-CBP mRNAs are expressed in the embryo. Interestingly, the results of immune electron microscopy showed that all three Arp-CBPs are distributed throughout the cellular ECL (embryonic cuticle layer) of the cyst shell. Furthermore, knockdown of Arp-CBP by RNA interference resulted in marked changes in the composition of the embryonic cuticular layer. The fibrous layer of the cyst shell adopted a loose conformation and the inner and outer cuticular membranes exhibited marked irregularities when Arp-CBP expression was suppressed. Finally, an in vitro recombinant protein-binding assay showed that all three Arp-CBPs have carbohydrate-binding activities. These findings provide significant insight into the mechanisms by which the ECL of Artemia cyst shell is formed, and demonstrate that Arp-CBPs are involved in construction of the fibrous lattice and are required for formation of the ECL of the cyst shell.